Treatment of cells of the promyeolocytic leukemia line, HL-60, with the phorbol ester, PMA, results in arrest of cell growth and induces terminal differentiation into monocytes. We have shown that in this response PMA initiates a rapid (less than 30 min.) and dramatic increase in phosphorylation of a 17kD cytosol protein (pp12). Although the phosphoester bond of this protein was highly alkalai stable, phosphoamino acid analysis revealed that the protein is not phosphorylated at tyrosine residues, but only at phosphoserine. Induction of phosphorylation of pp17 is intimately associated with the initial events by which PMA interacts with cells, since inactive phorbol esters--which do not bind to cellular binding sites--fail to induce the phosphorylation event. Moreover, increased phosphorylation of pp17 was also observed during the interaction of PMA with other cell types (U937, A431, and human lymphocytes). This suggests that induction of phosphorylation of pp17 may be a generalized initial response of cells to PMA treatment, promoting activation of the particular differentiation program of the cell lineage involved. The importance of this induction in activating the sequence of events leading to cellular differentiation is indicated by the failure of PMA to induce differentiation in the presence of trifluorperazine, which inhibits the PMA-induced phosphorylation of pp17. Kinetic studies showed that the phosphate group in pp17 is not stable in vivo, but undergoes rapid phosphorylation and dephosphorylation. Induction of phosphorylation of pp17 is not dependent on influx of extracellular calcium. Further characterization of the protein kinase involved in this reaction is in progress. Studies with methylation inhibitor, 3-deazaadenosine, indicate that the initial response of HL-60 cells to PMA, leading to induction of phosphorylation of pp17, does not require transmethylation reactions, although such reactions may play a role at a later time during differentiation.